Planta (1991)185:65-71
P l a n t ~ 9 Springer-Verlag1991
Riboflavin-binding sites associated with flagella of Euglena: A candidate for blue-light photoreceptor ? A. Nebenfiihr 1, A. Schiifer 1, P. Gailand 2, H. Senger 2, and R. Hertel 1. t Institut f'tir Biologie III der Universit~it, ScNinzlestrasse 1, W-7800 Freiburg i. Br. and 2 Fachbereich Biologie/Botanik, Universit~it Marburg, Lahnberge, W-3550 Marburg, Federal Republic of Germany Received 23 January; accepted 20 April 1991
Abstract. The hypothesis was tested that reversible riboflavin (RF)-binding sites are part of the photoreceptor in Euglena oracilis. Published evidence shows that the phototactic stimulus - with a flavin-type action spectrum - is perceived at the paraflagellar body (PFB). Flagella with PFBs were isolated from Euolena 9racilis by a combined cold and Ca ~+ shock. Saturable binding of [14C]RF was demonstrated with such preparations, in the oxidized state as well as under reducing conditions in the presence of dithionite. Affinities for R F were high: Ko (oxidized) = 0.08 gM, and KD (reduced) -: 0.7 gM. Flavin mononucleotide and flavin adenine dinucleotide showed lower binding affinities. The in vitro RF binding per unit of protein was enriched approximately tenfold in the flagellar preparations when compared with homogenates of whole cells. The number of (reduced) binding sites per entire flagellum was determined to be in the order of 1 0 6 . This number is in line with published estimates of chromophores bound in or at the PFB. Key words: Blue-light - Euolena - Flagellum - Flavin binding - Photoreceptor - Riboflavin
Introduction Well-defined blue-light photoresponses have been described for many organisms, but the relevant photoreceptor molecules are still unknown. The action spectra resemble the absorption spectra of flavins or carotenoids (Galland and Senger 1988). In some cases - e.g. Phy~ comyces phototropism (Presti et al. 1977) - the involvement of carotenoids can be ruled out; thus, flavins are considered to be the most likely photoreceptors. Most models of blue-light perception assume that the flavin chromophore is covalently or tightly bound to the protein moiety of the photoreceptor in the plasma mem* To whom correspondence should be addressed
Abbreviations: FAD=flavin adenine dinucleotide; FMN=flavin mononucleotide; PFB = paraflagellar body; RF = riboflavin
brane. In higher plants, however, the concentration of such tightly bound flavins seems to be too low to account for the observed physiological sensitivity (Hertel 1980). At the same time, the concentration of free cellular flavins (mostly riboflavin, RF) is relatively high, usually in the micromolar range. Hertel (1980) therefore proposed that the photoreceptor consists of a membrane protein and a "loosely" bound chromophore (RF) that is in exchange with a free, cytosolic pool. Indeed, a specific and reversible binding of R F to membrane preparations of Cucurbita, Zea (Hertel et al. 1980) and Phycomyces (Dohrmann 1983) has been observed. The model receives support from the work of Fritz et al. (1989) on Neurospora mutants deficient in R F biosynthesis. Low concentrations (0.08 gM) of R F added to the medium allowed hyphal growth, but photoresponses here, phase shifts of the circadian clock - were still suppressed. Light sensitivity developed only at higher RF concentrations, above 0.14 gM in the medium; it further increased with increasing extracellular RF concentrations up to 20 gM, corresponding to approx. 8 gM intracellular RF. Several chemical RF analogs were tested that cannot be converted to flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD), or covalently attached to proteins. These analogs showed essentially the same "photo-sensitizing" effect as did RF. Analogs, however, that could not penetrate the cell surface were not able to support any light response. Fritz et al. (1989) therefore concluded that "free cellular R F acts as the photoreceptor for light-induced phase shifts of the circadian conidiation rhythm in Neurospora". Up to the present, no causal biochemical connection between a reversible binding of R F and any photophysiological response has been shown. It is possible, however, to approach this question indirectly by using an organism in which the subcellular localization of the photoreceptor is precisely known, and testing whether in vitro RF-binding sites are particularly enriched in this location, e.g. in a specific organelle. In the case of Euglena 9raeilis, the photoreceptor is located in or at the paraflagellar body (PFB) of the major flagellum (see Kuznicki et al. 1990). The stigma ("eye-
66 spot") is excluded as the p h o t o r e c e i v i n g organelle because cells bleached with s t r e p t o m y c i n lose their chloroplast a n d stigma, b u t nevertheless retain p h o t o t a x i s (Checcucci et al. 1976). F u r t h e r experiments have s h o w n that w h e n a laser m i c r o b e a m is directed only to the area o f the P F B , a n d n o t to o t h e r parts o f the Euglena cell, there occurs a v i o l e n t stroke r e a c t i o n o f the flagellum ( C o l o m b e t t i et al. 1982). F l a v i n - t y p e a u t o f l u o r e s c e n c e has been reported to occur in the P F B region o f intact, living Euglena (Benedetti a n d Checcucci 1975) a n d o f isolated flagella ( S c h m i d t et al. 1990). I n a d d i t i o n , flavoproteins have been d e m o n s t r a t e d in flagellar p r e p a r a t i o n s by B r o d h u n a n d H/ider (1990). It s h o u l d be m e n t i o n e d that pterins are discussed as possible auxiliary p h o t o r e c e p t o r s at the flagellar base ( B r o d h u n a n d H~ider 1990; G a l l a n d et al. 1990; S c h m i d t et al. 1990). G u a l t i e r i et al. (1986) devised a m e t h o d to isolate the m a j o r flagellum o f Euglena gracilis with the P F B still attached. I f R F - b i n d i n g sites are actually associated with p h o t o r e c e p t i o n they s h o u l d be f o u n d in high c o n c e n t r a tion in such p r e p a r a t i o n s . T h e e x p e r i m e n t s reported here test this prediction.
Materials and methods
Radiochemicals and chemicals. [2-14C]Riboflavin (2 GBq" mmole-i) was purchased from Amersham Buchler (Braunschweig, FRG); c~/fl-octylglucosidefrom Sigma (Mfinchen, FRG) and from Biomol (Hamburg, FRG). Mouse monoclonal anti-~-tubulin antibodies, clone DM1A, and anti-mouse IgG, alkaline-phosphatase conjugate, were purchased from Sigma where the other materials for gels and blotting were also obtained. All other chemicals were obtained from Merck (Darmstadt, FRG) and Roth (Karlsruhe, FRG).
Growth conditions. A culture of Euglena #racilis, strain Z Pringsheim, obtained from the culture collection in G6ttingen, FRG (Schl6sser 1982) was inoculated in 2 or 51 carboys containing culture medium described by Beale et al. (1981). The cells were grown as described by Galland et al. (1990) in a controlled culture room at 22~ under continuous illumination from fluorescent lamps (3 W - m-Z). A current of air was filtered through several stages of sterile cotton or glass-wool filters, and bubbled through the cultures. The growth period lasted some 8 d until the cultures had reached a density of approx. 106 cells-ml -a.
Isolation procedure. The flagella were isolated according to the method of Gualtieri et al. (1986) with modifications by Galland et al. (1990). In a first low-speed centrifugation (1000 - 9 for 10 rain) at room temperature, the cells were pelleted and then resuspended at a titer of about 107 cells per ml. (Higher accelerations or chilling cause premature deflagellation.) All subsequent steps were carried out at 4 ~ C or on ice. Calcium chloride was added to a final concentration of 100 mM to detach the flagella which were then separated by a second lowspeed centrifugation (1000 9g for 10 min at 4 ~ C) to sediment the cells, and finally pelleted at 22 000 9g for 20 min. The flagella were resuspended in 2 ml binding-assay buffer (10 mM Tris-HC1, 5 mM MgSO4, pH 7.5), blended in a glass homogenizer and frozen at -80~ for later tests. Freezing did not affect the binding capacity. Cells were homogenized by grinding them to powder under liquid N 2 in a mortar. In one experiment (see Fig. 4), the cell homogenate was prepared by treating the material, frozen in liquid N2, for 6 - 30 s in a "MikroDismembrator II" (from Braun, Melsungen, FRG).
A. Nebenfiihr et al. : Riboflavin-bindingto Euglena flagella At all stages of the isolation, cells and-or flagella were counted under a light microscope (Type III RS; Zeiss, Oberkochen, FRG). The final yield was low, usually about 5% of the flagella present in the original culture.
Binding assay. The binding test - modified according to Hertel et al. (1980) - was routinely performed in 1.5-ml micro centrifuge tubes (Eppendorf, Hamburg, FRG) under normal laboratory light. Test buffer (10 mM Tris-HC1, 5 mM MgSO4, pH 7.5, with 0.1 or 0.2% octylglucoside), [i4C]RF (often 5. 10 -8 M) and varying amounts of unlabelled RF were added as specified. The detergent was added to facilitate ligand access. If the binding properties of reduced RF were to be investigated, 5 mM Na-dithionite was added quickly from 125 mM dithionite dissolved in H20 immediately before use. The assay was started with the addition of 100 lal of the Euglena material. The final volume was 0.5 ml per test. After an incubation period of 10 rain on ice, the total volume was diluted in 5 ml of 10 mM Tris-HC1 (pH 7.5) and immediately poured over glass-fiber filters (Whatman GF/B; Bender und Hobein, Freiburg, FRG) into a vacuum flask; the filters had been preincubated in 0.3% polyethyleneimine (PEI) for 1 h (filter test modified after Thein and Michalke 1988). To avoid unspecific attachment of radioactivity to the filters they were rinsed with another 5 ml of 10 mM Tris-HC1 (pH 7.5). The PEI-coated filters, where the protein-RF-complex is retained, were clear of liquid about 10 s after dilution, and were then transferred to scintillation fluid (Ultima Gold from Beckmann Co, Mfinchen, FRG) and counted in a Beckmann Liquid Scintillation Counter. 'Saturable' or 'specific' [14C]RF binding is defined as the difference in filter-bound radioactivity (in cpm) between a sample containing only radioactive RF and a parallel sample to which saturating amounts of unlabelled RF had been added. Binding is often expressed as % binding=saturably bound cpm- 100/total cpm in assay. This value per mg protein added to the assay, can be used to compare different preparations. Routinely, assays were run in duplicate; the mean is presented. Table 1 illustrates the raw data and the unspecific binding: e.g. the duplicate samples for the left column were 85 and 75 cpm, or 13 and 35 cpm, respectively, and for the right column 212 and 214 cpm, or 14 and 13 cpm, respectively. This gives an impression of the accuracy of the binding tests. Most other data are presented as "specific (= saturable) binding".
Polyacrylamide 9el electrophores&, Western blots and prote& determination. Samples from Euglena cells and flagella were separated on a 10% acrylamide sodium dodecyl sulfate gel according to Laemmli (1970). One half of the gel was then transferred to a semi-dry blotting apparatus (Biometra, G6ttingen, FRG); the proteins were blotted electrophoretically to a nitrocellulose membrane (BA 83, 0.2 ~tm; Schleicher und Schiill, Dassel, FRG). Positive bands were detected using mouse monoclonal anti-a-tubulinantibodies (1/2000 in test) and enzyme-conjugated anti-mouse-IgG (1/3000), and the blot was stained with 5-bromo-4-chloro-3-indolyl phosphate, ptoluidine salt/nitro blue tetrazolium (BCJP/NBT). Western blotting and staining according to Harlow and Lane (1988). For protein determination the Coomassie method (Spector 1978) was used.
Results
Riboflavin binding in flagellar preparations. The first a i m was to detect a n d characterize s a t u r a b l e b i n d i n g o f [14C]RF in flagellar m a t e r i a l isolated a c c o r d i n g to G u a l tieri et al. (1986). This p r o c e d u r e e m p l o y s a c a l c i u m shock that causes the flagella to beat vigorously, t h e r e b y everting the a n t e r i o r i n v a g i n a t i o n in which the base o f the flagellum is attached. This ejection shifts the p o i n t o f
A. Nebenfiihr et al. : Riboflavin-binding to Euglena flagella highest stress towards the flagellar base, just above the cell membrane. As a consequence, flagella break off below the PEB which remains attached to its flagellum. Thus, isolation of flagella by differential centrifugation will co-purify PFBs and separate them from the cell bodies. During the procedure an enrichment of flagellar structures and of tubulin (see below, Fig. 4) was consistently observed, The flagellar structures counted under the light microscope were of very variable length. Either they broke off at different points outside the invagination, or they were broken during homogenization. Therefore, one has to assume that not all o f the flagellar pieces counted probably less than 50% - carried a P F B . . ~ The results in Table 1 show that both oxidized and reduced R F can bind to the flagella isolated as described above. In the flagellar preparations, p4C]RF binding was proportional to protein concentration, at least between 0 and 60 gg (data not shown). Dithionite indeed acts as a reducing agent. The products o f its oxidation, e.g. bisulfite, do not interfere with the test, since R F remains in the reduced state which is not attacked by bisulfite (Miiller and Massey 1969). After 40 min incubation of the dithionite test mixture, reoxidation had occurred as seen by the reappearance o f oxidized (yellow) RF. The R F binding, both in the oxidized and the reduced state, was reversible since radioactivity could be displaced f r o m the sites after the binding incubation, by adding a surplus of unlabelled R F to the dilution medium and leaving the diluted samples for 10 min (data not shown). Saturation curves with increasing R F concentrations are presented in Fig. la. F r o m these data and f r o m the corresponding Scatchard plots (Fig. lb), affinities and number o f sites can be estimated. In the absence of dithionite, the n u m b e r of "oxidized" binding sites (Fig. 1b, open symbols, intersection of the regression line with the abscissa: 3.2 pmol per assay, corresponding to 0.05 nmol 9 m g - 1 protein), and their affinity towards the ligand (Fig. l b, negative reciprocal of the slope, a K D --- 0.07 laM) were calculated. In another similar experiment these values were 0.8 pmol and 0.09 gM, respectively. In the presence of dithionite, the n u m b e r o f binding sites (Fig. lb, closed symbols) was determined as 7 pmol
67 a 120
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20
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+Unlabelled RF Saturable binding % bound/free RF % bound/free, mg -1 protein
Oxidized
Reduced
80 cpm 24 cpm 56 cpm 3.0 75.3
214 cpm 14 cpm 200 cpm 11 274
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O.02J Table 1. p4C]Riboflavin binding to a flagellar preparation from Euglena in the presence (reduced) and in the absence (oxidized) of dithionite. 40 gg protein or approx. 4 9107 flagella were used in each sample. The radioactive ligand had a concentration of 4- 10 -s M [14C]RF (i.e. 1861 cpm); 3 9 10-5 M unlabelled RF was added to part of the samples as competitor. Each value is based on duplicate determinations
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o 5
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~ 10
["C]-Riboflavin b o u n d
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Fig. 1. a Saturation curves at increasing RF concentrations under oxidizing conditions (open symbols), using 60 gg of protein (approx. 6 - 107 flagella) in each test sample, and under reducing conditions (5 mM dithionite added; closed symbols), using 6.9 gg protein (4.2 - 106 flagella) b Scatchard plots calculated from data in a under oxidizing conditions (open symbols), and under reducing conditions
(closed symbols)
A. Nebenfiihr et al. : Riboflavin-bindingto Euglena flagella
68
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underestimated because the flavins present in vivo might still be occupying some of the sites.
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Fig. 2. Saturation curves with increasing concentrations of R F (squares), F M N (triangles), and F A D (diamonds). Each sample contained 40 lag protein, approx. 4 - 106 flagella. To this was added 4.1 9 10 -8 M (i.e. 2000 cpm) [14C]RF and varying concentrations of unlabelled flavins, but no dithionite. The values at 10-7 M (filled symbols) represent the means of four replicates each; here, the standard error were about the size of the symbols (___2 cpm)
per assay (corresponding to 1.0 nmol. mg -1 protein), and their R F affinity was characterized by a dissociation constant K[, of 0.7 rtM. Centrifuge binding assays (according to Hertel et al. 1980) produced essentially the same results as did the filter tests (data not shown). The binding tests were usually performed in laboratory daylight. Control experiments under red safelight did not reveal any difference from those under the standard conditions (data not shown), i.e. there was no effect of light on RF binding. To determine whether RF was a specific ligand for these sites, increasing concentrations of some analogs were tested for their capacity to compete with [x4C]RF (Fig. 2). When tested in the absence of dithionite, FAD showed a much lower affinity for the binding sites, whereas F M N was more effective in replacing p4C]RF. However, it still had significantly less affinity than RF itself, by a factor of approx. 2. The same pattern was obtained in the reduced state; substances like xanthopterin and indoleacetic acid did not compete at all (data not shown).
Estimate of number of binding sites per flagellum. The molarity of RF-binding sites in the assays can be converted to binding sites per protein or - more relevantly - the number of binding sites per flagellar piece counted. In experiments without dithionite approx. 3" 104 sites per flagellum are estimated. In the case of the reduced ligands (see Fig. lb, closed symbols), however, the number of binding sites seems to be much higher, approx. 5 9 105 per flagellum. (It should be noted that in six separate and independent experiments, the estimated number of binding sites per flagellum varied by a factor of 2.8.) The estimates of binding sites represent minimal values only because the number of "entire" flagella may be overestimated. On the other hand, the number of sites could be
Localization of binding sites. The main experimental aim was to compare flagella and cell bodies of Euglena with respect to RF binding. The isolation procedure according to Gualtieri et al. (1986), separates flagella with intact PFBs from the cells; thus, the presumed photoreceptor and - according to our hypothesis - the RF-binding sites should be highly enriched in flagella compared to whole cells. In numerous experiments, using independent preparations, and under various conditions, the RF-binding capacity of isolated flagella as well as that of the cells, was assayed. The most striking and important qualitative feature in all independent tests was the high enrichment of in vitro RF binding per unit of protein in the flagella when compared to cells. Saturation curves of RF binding to flagella and to deflagellated cells were compared under oxidizing conditions (Fig. 3). The absence of binding capacity in the cell homogenate might be attributed to the action of substances present in this crude preparation and inhibiting RF binding. However, the possibility of such an inhibitor was ruled out since a mixture of the isolated flagella and the cell homogenate showed exactly the same binding capacities as did the flagella alone (Fig. 3). For reducing conditions, the same difference between flagella and cells was found (Table 2: one experiment in detail; Table 3: summary of three tests). In these experiments (Tables 2, 3), isolated flagella were compared to a homogenate of still-flagellated cells. These cells did show RF-binding capacity which was small on a protein basis, but the numbers per cell are similar to those per flagellum (Table 2, last line). Again, the strong enrichment of binding per protein in flagella cannot be attributed to inhibitor(s) in the cell 90 80~-~ 70(D
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Fig. 3. Riboflavin binding to flagella and cell homogenates of Euglena, and mixtures thereof. The flagella and cell samples contained 30 gg and 40 lag protein, respectively. The mixtures were simple additions of 30 lag flagellar and of 40 gg cell protein. Other conditions were as in Fig. 2. (D-[] flagella, V-V cell homogenate, /x-/x flagella + cell homogenate)
A. Nebenfiihr et al. : Riboflavin-binding to Euglena flagella
69
Table 2. Binding of R F to isolated flagella and cell homogenates of Euglena and to a mixture thereof. 6 . 5 - 1 0 -8 M (i.e. 3048cpm) [14C]RF 4-3 - 10 -5 M unlabelled R F was added. All samples contained 5 m M Na-dithionite to achieve reducing conditions. 'Pieces' are either ceils, or flagella, or fragments of flagella counted in the microscope. Numbers in parentheses are extrapolated for the mixture Homogenate Flagella from cells + homogenate from cells
Flagella
Protein (pg) N u m b e r of pieces
6.0 4.9 9 106
cpm spec. bound % bound/free R F % b o u n d / f r e e - m g -x protein Binding sites/piece
67.4 2.6 9 106
(73.40) (7.5 9 106)
29.0
26.9
51.2
0.95 158.6
0.88 13.1
1.68 22.9
4.3 - 105
7.4" l0 s
(4.9" 105)
Table 3. Binding of R F to flagella and cell homogenates of Euglena and to mixtures thereof: three independent preparations. The cells used still carried their flagella. The protein concentration of the cell homogenate was usually about ten times that of the flagellar sample. All samples contained 5 m M Na-dithionite to obtain reducing conditions. The values are given as '% b o u n d / f r e e ' m g -1 protein'. Other conditions as in Table 2 Expt. No.
R F binding in flagella
1 2 3
159 171 154
homogenates from cells
fagella + homogenates from cells
13 8 11
23 18 13
where flagella had been shaken off (Table 4). As another control, a "flagellar" preparation was included which was derived from cells that were previously deprived of their flagella; its binding capacity was very low (Table 4, third column). In some experiments the flagellar content of the preparations was checked by immunodetection of tubulin in the different samples. For this we employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots with subsequent incubation with anti-tubulin antibodies. Euglena tubulin antigen appeared at the expected molecular weight of 55 kDa, and co-migrated with known bovine brain tubulin (data not shown). Figure 4 demonstrates the relative abundance of tubulin in the flagellar preparation compared to the cells. The RF binding (oxidized state) in these two fractions was determined to be 16.2% 9rag- 1 protein for flagella, and 2.0 % 9mg-1 protein for cells. The result confirms the microscopic estimates and again shows that RF binding is enriched in the flagella. In this experiment, the cells were homogenized with an additional vigorous treatment in a "micro-dismembrator". The resulting extract was taken up in nine volumes of test buffer, and large pieces and cells were removed by a centrifugation at 1300"9 for 10 min. This preparation showed the same binding properties as did the standard cell homogenate used in the other experiments.
Riboflavin binding to flagellar preparations and their supernatant. During this study, it was found that the
homogenate because the absolute amount of saturably bound radioactivity in the mixture of flagella and cells is the sum of the values for flagella, and for cells alone (Table 2). It should be noted that the binding values per unit of protein in Tables 2 and 3 are low for the mixtures because binding sites are "diluted" by the large amount of cell protein added. It was also found that homogenates from intact Eug~ lena cells display better RF binding than those from cells
supernatant obtained from a flagellar preparation contained an appreciable number of RF-binding sites. Flagella were sedimented at the intermediate centrifugal force of 22 000"g (characterization and R F binding shown in Table 5, left column); the resulting supernatant was subjected to a further, high-speed centrifugation at 200 000"g for 40 min. This final pellet showed RF binding (Table 5, right column), and it contained flagella, but in numbers too small to account for the large amount of binding observed. If, during preparation of flagella, some PFBs - much smaller than flagella - were broken off they might be left in the supernatant of the centrifugate at 22 000 9g, sedimenting, however, at the higher force.
Table 4. Binding of R F in various flagellar and cellular preparations. Deflagellated Euglena cells were obtained as described in Materials and methods. The 'flagellar' preparation in the third colu m n was derived from previously deflagellated cells by a second Ca 2+ shock, corresponding to fraction III in Galland
et al. (1990), (Fig. 1 .). All samples contained 40 lag protein, except for that in the third column with only 18 lag, and a very low number of flagella. No dithionite was added. Each sample contained 3 9 10 -8 M [14C]RF; 3 - 10 -5 M unlabelled R F was added to half of the samples
Starting from whole cells :
deflagellated cells:
flagella
homogenate
'flagella'
homogenate
+ Unlabelled R F Saturable binding
94 cpm 22 cpm 72 cpm
58 cpm 26 cpm 32 cpm
34 cpm 23 cpm 11 cpm
32 cpm 20 cpm 12 cpm
% bound/free R F % bound/free 9 mg -1 protein
3.9 96.3
1.7 42.8
0.6 32.7
0.6 16.0
A. Nebenfiihr et al. : Riboflavin-binding to Euglenaflagella
70 M
F5
FIO
C5
C10
M
ii !! ii i~i
55 k D a >
Table 5. Binding of RF to isolated flagella of Euglena and to the high-speed pellet from a 'flagellar' supernatant (centrifugation at 200000 9g for 40 min). The [14C]RF concentration was 7 9 10 -8 M. All samples contained 5 m M Na-dithionite. The number of binding sites was calculated assuming a KD of 0.7 taM
Protein (gg) Number of flagella Saturable binding (cpm) Bound/free 9mg -l protein (%) Binding sites in assay Binding sites/flagellum
Flagella
Pelleted supernatant
6.9 4.2 l06 39.2 170.8 2.6.1012 6.2.105
6.9 1.6 " 106 39.3 171.0 2.6.1011 1.6- 106
'
Furthermore, preliminary tests indicate that flagella without a PFB bind much less RF. The isolated flagella had been subjected to an additional Ca 2 + shock. After a subsequent centrifugation, according to Brodhun and Hfider (1990), the pelleted flagella should contain no PFBs. In parallel assays, both regularly prepared flagella and flagella with their PFBs removed were exposed to [14C]RF: in the oxidized state, the latter showed a saturable R F binding (as bound/free 9 m g - 1 protein) of only 0.21 as compared to 0.96 for control flagella. M
F5
FIO
C5
C10
M
Discussion
Fig. 4. a Analysis of flagella and cell preparations by SDS PAGE,
and b subsequent Western blot with anti-a-tubulin antibodies. The proteins in a and b were separated on one common 10% gel which subsequently was cut into two halves for protein staining and blotting, respectively. The lanes were loaded with 5 tag and 10 tag protein, respectively, as indicated above each lane. F, flagellar preparation; C, cell extract; M, molecular-weight markers (prestained markers from Sigma, FRG)
Proteins, in the plasma m e m b r a n e or in another "fixed" position, that bind R F have been proposed as blue-light photoreceptors in fungi and plants (Hertel 1980). The results here document the occurrence of a strong and saturable binding of R F to flagellar preparations from Euglena. Both oxidized and reduced R F can bind specifically, i.e. better than F M N or F A D . Affinity for reduced as well as for oxidized R F was reflected by Ko values in the range of 0.1 - 1 gM. The number of RF-binding sites seems to be higher in the presence of dithionite. The difference between the number o f sites in the reduced and the oxidized state suggests that one is dealing with two different types of binding proteins. The decision as to whether there are two or more different binding sites involved, or just different affinity patterns at one site only, must await isolation and characterization of the respective proteins. Such a study is presently under way. F r o m the a m o u n t of R F binding and from the number of flagella (or flagellar pieces) in the assay, approx. 5" 105 RF-binding sites per flagellum were estimated under reducing conditions. The amount per PFB might actually be higher by a factor of two, or more, since probably only half of the "flagella" counted carried their PFB. The number of 106 binding sites per PFB should be compared to: (i) the a m o u n t of binding sites from one intact cell (Table 2); (ii) the number of photoreceptor molecules in the PFB that can be expected from microspectrophotometric observations (Colombetti and Lenci 1980); (iii) the number of flavin molecules in/at the PFB determined by Colombetti and Lenci (1980) by microspectrofluorimetry; and (iv) the number of unit cells o f the PFB's protein crystal (Piccini and M a m m i 1978).
A. Nebenfiihr et al. : Riboflavin-binding to Euglena flagella Since all these n u m b e r s are in the range o f approx. 106, it seems reasonable to p r o p o s e that the R F - b i n d i n g sites are localized in the P F B or at the s u r r o u n d i n g m e m b r a n e , and that these proteins are involved in p h o t o r e c e p t i o n for euglenoid phototaxis. It has been observed that the flavin-type fluorescence in the P F B region o f Euglena described by Benedetti and Checcucci (1975) a n d b y Schmidt et al. (1990) is lost when preparing and washing flagella (Galland et al. 1990, fraction II in their Fig. 1). These findings agree very well with a "loose" flavin binding to the flagellar structures. The finding by B r o d h u n and H/ider (1990) o f some covalently (or tightly) b o u n d flavin in flagellar preparations is n o t incompatible with the large a m o u n t o f reversible R F binding s h o w n here. Gualtieri et al. (1989) m e a s u r e d a b s o r p t i o n spectra f r o m a single "dried" P F B in situ. The a m o u n t o f pigment m u s t be very high, in the range o f 107 molecules per PFB. The interpretation o f the spectrum as being that o f a r h o d o p s i n is n o t in line with the reported action spectra (see Introduction). We rather suggest that P F B s contain a protein-stabilized " r e d " flavin semiquinone radical (see Massey a n d Palmer 1966, where e.g. D-amino acid oxidase shows a spectrum like the one m e a s u r e d by G u a l tieri et al. 1989). The pterins - p r o b a b l y present at the P F B - m a y act as a second p h o t o r e c e p t o r , or as a c o f a c t o r for the p h o t o reactions ( B r o d h u n a n d H~ider 1990; G a l l a n d et al. 1990; Schmidt et al. 1990). Interference o f pterins with R F binding, however, was n o t seen when x a n t h o p t e r i n was added. The determination o f the m e c h a n i s m o f p h o t o p e r c e p tion in Euglena is o f general interest because this p r o t o zoon/phytoflagellate m a y be at the base o f eukaryotic evolution. Euglena's - a n d m o s t protists - precise phylogenetic relationships are far f r o m clear, but it m i g h t actually represent a p r o t o z o o n that carries a e u k a r y o t i c alga as an e n d o s y m b i o n t . F o r example, its pellicle lacking cellulose is atypical o f cell walls o f green algae, and the "chloroplasts" have one additional m e m b r a n e which has been interpreted as the r e m n a n t o f an e n d o c y t o t i c event in which a e u k a r y o t i c green alga was i n c o r p o r a t e d into the pre-Euglena cell (Gibbs 1978). In view o f these relationships it is m o s t interesting that other non-green algae also s h o w a strong flavin-like fluorescence in one o f their flagella (Kawai 1988; Colem a n 1988). Riboflavin-binding sites in these flagella w o u l d be a n o t h e r strong a r g u m e n t for a p h o t o p h y s i o l o g ical role o f such flavoproteins. It should be noted, h o w ever, that in Euglena, flavin fluorescence o f the flagellum - in c o n t r a s t to the P F B - is very weak (Kawai 1988). This work was supported by the Deutsche Forschungsgemeinschaft
References Beale, S.I., Foley, T., Dzelzkalns, V. (1981) 8-Aminolevulinic acid synthase from Euglena graeilis. Proc. Natl. Acad. Sci. USA 78, 1666-1669 Benedetti, P.A., Checcucci, A. (1975) Paraflagellar body (PFB) pigments studied by fluorescence microscopy in Euglena graeilis. Plant Sci. Letts. 4, 47-51 Brodhun, B., H~ider, D.P. (1990) Photoreceptor proteins and pig-
71 ments in the paraflagellar body of the flagellate Euglena 9racilis. Photochem. Photobiol. 52, 865-872 Checcucci, A., Colombetti, G., Ferrara, R., Lenci, F. (1976) Action spectra for photoaccumulation of green and colorless Euglena: evidence for identification of receptor pigments. Photochem. Photobiol. 23, 51-54 Coleman, A.W. (1988) The autofluorescent flagellum: a new phylogenetic enigma. J. Phycol. 24, 118-120 Colombetti, G., Lenci, F. (1980) Identification and spectroscopic characterization of photoreceptor pigments. In: Photoreception and sensory transduction in aneural organisms (NATO ASISeries A 33), pp. 173-188, Lenci, F., Colombetti, G., eds. Plenum Press, New York Colombetti, G., Lenci, F., Diehn, B. (1982) Responses to photic, chemical, and mechanical stimuli. In: The biology of Euglena III. pp. 169-196, Buetow, D.E., ed. Academic Press, New York Dohrmann, U. (1983) In vitro riboflavin binding and endogenous flavins in Phycomyces blakesleeanus. Planta 159, 357-365 Fritz, B.J., Kasai, S., Matsui, K. (1989) Free cellular riboflavin is involved in phase shifting by light of the circadian clock in Neurospora crassa. Plant Cell Physiol. 30, 557-564 Galland, P., Senger, H. (1988) New trends in photobiology: The role of flavins as photoreceptors. J. Photochem. Photobiol. B 1, 277-294 Galland, P., Keiner, P., D6rnemann, D., Senger, H., Brodhun, B., H~ider, D.P. (1990) Pterin- and flavin-like fluorescence associated with isolated flagella of Euglena 9racilis. Photochem. Photobiol. 51, 675-680 Gibbs, S.P. (1978) The chloroplasts of Euglena may have evolved from symbiotic green algae. Can. J. Bot. 56, 2883-2889 Gualtieri, P., Barsanti, L., Rosati, G. (1986) Isolation of the photoreceptor (paraflagellar body) of the phototactic flagellate Euglena 9racilis. Arch. Microbiol. 145, 303-305 Gualtieri, P., Barsanti, L., Passarelli, V. (1989) Absorption spectrum of a single paraflagellar swelling of Euglena 9racilis. Biochim. Biophys. Acta 993, 293-296 Harlow, E., Lane, D. (1988) Antibodies: a laboratory manual. Cold Spring Harbor Lab., New York Hertel, R. (1980) Phototropism in lower plants. In : Photoreception and sensory transduction in aneural organisms (NATO ASISeries A 33), pp, 89-105, Lenci, F., Colombetti, G., eds. Plenum Press, New York Hertel, R., Jesaitis, A.J., Dohrmann, U., Briggs, W.R. (1980) In vitro binding of riboflavin to subcellular particles from maize coleoptiles and Cucurbita hypocotyls. Planta 147, 312-319 Kawai, H (1988) A flavin-like autofluorescent substance in the posterior flagellum of golden and brown algae. J. Phycol. 24, 114-117 Kuznicki, L., Mikolajzcyk, E., Walne, P.L. (1990) Photobehavior of euglenoid flagellates: theoretical and evolutionary perspectives. Crit. Rev. Plant Sci. 9, 343-369 Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680-685 Massey, V., Palmer, G. (1966) On the existence of spectrally distinct classes of flavoprotein semiquinones. Biochemistry 5, 3181-3189 Miiller, F., Massey, V. (1969) Flavin-sulfite complexes and their structures. J. Biol. Chem. 224, 4007-4016 Piccini, E., Mammi, M. (1978) Motor apparatus ofEuglena 9racilis: ultrastructure of the basal portion of the flagellum and the paraflagellar body. Boll. Zool. 45, 405-414 Presti, D.E., Hsu, W.J., Delbriick, M. (1977) Phototropism in Phyeomyees mutants lacking [3-carotene. Photochem. Photobiol. 26, 403-405 Schl6sser, U.G. (1982) Sammlung von Algenkulturen. Ber. Dtsch. Bot. Ges. 95, 181-276 Schmidt, W., Galland, P., Senger, H., Furuya, M. (1990) Microspectrophotometry of Euglena gracilis. Pterin- and flavin-like fluorescence in the paraflagellar body. Planta 182, 374-381 Spector, T. (1978) Refinement of the Coomassie-Blue-method of protein quantitation. Annal. Biochem. 86, 142-146 Thein, M., Michalke, W. (1988) Bisulfite interacts specifically with binding sites of the auxin transport inhibitor N-1-Naphthylphthalamic acid. Planta 76, 343 350
P l a n t ~ 9 Springer-Verlag1991
Riboflavin-binding sites associated with flagella of Euglena: A candidate for blue-light photoreceptor ? A. Nebenfiihr 1, A. Schiifer 1, P. Gailand 2, H. Senger 2, and R. Hertel 1. t Institut f'tir Biologie III der Universit~it, ScNinzlestrasse 1, W-7800 Freiburg i. Br. and 2 Fachbereich Biologie/Botanik, Universit~it Marburg, Lahnberge, W-3550 Marburg, Federal Republic of Germany Received 23 January; accepted 20 April 1991
Abstract. The hypothesis was tested that reversible riboflavin (RF)-binding sites are part of the photoreceptor in Euglena oracilis. Published evidence shows that the phototactic stimulus - with a flavin-type action spectrum - is perceived at the paraflagellar body (PFB). Flagella with PFBs were isolated from Euolena 9racilis by a combined cold and Ca ~+ shock. Saturable binding of [14C]RF was demonstrated with such preparations, in the oxidized state as well as under reducing conditions in the presence of dithionite. Affinities for R F were high: Ko (oxidized) = 0.08 gM, and KD (reduced) -: 0.7 gM. Flavin mononucleotide and flavin adenine dinucleotide showed lower binding affinities. The in vitro RF binding per unit of protein was enriched approximately tenfold in the flagellar preparations when compared with homogenates of whole cells. The number of (reduced) binding sites per entire flagellum was determined to be in the order of 1 0 6 . This number is in line with published estimates of chromophores bound in or at the PFB. Key words: Blue-light - Euolena - Flagellum - Flavin binding - Photoreceptor - Riboflavin
Introduction Well-defined blue-light photoresponses have been described for many organisms, but the relevant photoreceptor molecules are still unknown. The action spectra resemble the absorption spectra of flavins or carotenoids (Galland and Senger 1988). In some cases - e.g. Phy~ comyces phototropism (Presti et al. 1977) - the involvement of carotenoids can be ruled out; thus, flavins are considered to be the most likely photoreceptors. Most models of blue-light perception assume that the flavin chromophore is covalently or tightly bound to the protein moiety of the photoreceptor in the plasma mem* To whom correspondence should be addressed
Abbreviations: FAD=flavin adenine dinucleotide; FMN=flavin mononucleotide; PFB = paraflagellar body; RF = riboflavin
brane. In higher plants, however, the concentration of such tightly bound flavins seems to be too low to account for the observed physiological sensitivity (Hertel 1980). At the same time, the concentration of free cellular flavins (mostly riboflavin, RF) is relatively high, usually in the micromolar range. Hertel (1980) therefore proposed that the photoreceptor consists of a membrane protein and a "loosely" bound chromophore (RF) that is in exchange with a free, cytosolic pool. Indeed, a specific and reversible binding of R F to membrane preparations of Cucurbita, Zea (Hertel et al. 1980) and Phycomyces (Dohrmann 1983) has been observed. The model receives support from the work of Fritz et al. (1989) on Neurospora mutants deficient in R F biosynthesis. Low concentrations (0.08 gM) of R F added to the medium allowed hyphal growth, but photoresponses here, phase shifts of the circadian clock - were still suppressed. Light sensitivity developed only at higher RF concentrations, above 0.14 gM in the medium; it further increased with increasing extracellular RF concentrations up to 20 gM, corresponding to approx. 8 gM intracellular RF. Several chemical RF analogs were tested that cannot be converted to flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD), or covalently attached to proteins. These analogs showed essentially the same "photo-sensitizing" effect as did RF. Analogs, however, that could not penetrate the cell surface were not able to support any light response. Fritz et al. (1989) therefore concluded that "free cellular R F acts as the photoreceptor for light-induced phase shifts of the circadian conidiation rhythm in Neurospora". Up to the present, no causal biochemical connection between a reversible binding of R F and any photophysiological response has been shown. It is possible, however, to approach this question indirectly by using an organism in which the subcellular localization of the photoreceptor is precisely known, and testing whether in vitro RF-binding sites are particularly enriched in this location, e.g. in a specific organelle. In the case of Euglena 9raeilis, the photoreceptor is located in or at the paraflagellar body (PFB) of the major flagellum (see Kuznicki et al. 1990). The stigma ("eye-
66 spot") is excluded as the p h o t o r e c e i v i n g organelle because cells bleached with s t r e p t o m y c i n lose their chloroplast a n d stigma, b u t nevertheless retain p h o t o t a x i s (Checcucci et al. 1976). F u r t h e r experiments have s h o w n that w h e n a laser m i c r o b e a m is directed only to the area o f the P F B , a n d n o t to o t h e r parts o f the Euglena cell, there occurs a v i o l e n t stroke r e a c t i o n o f the flagellum ( C o l o m b e t t i et al. 1982). F l a v i n - t y p e a u t o f l u o r e s c e n c e has been reported to occur in the P F B region o f intact, living Euglena (Benedetti a n d Checcucci 1975) a n d o f isolated flagella ( S c h m i d t et al. 1990). I n a d d i t i o n , flavoproteins have been d e m o n s t r a t e d in flagellar p r e p a r a t i o n s by B r o d h u n a n d H/ider (1990). It s h o u l d be m e n t i o n e d that pterins are discussed as possible auxiliary p h o t o r e c e p t o r s at the flagellar base ( B r o d h u n a n d H~ider 1990; G a l l a n d et al. 1990; S c h m i d t et al. 1990). G u a l t i e r i et al. (1986) devised a m e t h o d to isolate the m a j o r flagellum o f Euglena gracilis with the P F B still attached. I f R F - b i n d i n g sites are actually associated with p h o t o r e c e p t i o n they s h o u l d be f o u n d in high c o n c e n t r a tion in such p r e p a r a t i o n s . T h e e x p e r i m e n t s reported here test this prediction.
Materials and methods
Radiochemicals and chemicals. [2-14C]Riboflavin (2 GBq" mmole-i) was purchased from Amersham Buchler (Braunschweig, FRG); c~/fl-octylglucosidefrom Sigma (Mfinchen, FRG) and from Biomol (Hamburg, FRG). Mouse monoclonal anti-~-tubulin antibodies, clone DM1A, and anti-mouse IgG, alkaline-phosphatase conjugate, were purchased from Sigma where the other materials for gels and blotting were also obtained. All other chemicals were obtained from Merck (Darmstadt, FRG) and Roth (Karlsruhe, FRG).
Growth conditions. A culture of Euglena #racilis, strain Z Pringsheim, obtained from the culture collection in G6ttingen, FRG (Schl6sser 1982) was inoculated in 2 or 51 carboys containing culture medium described by Beale et al. (1981). The cells were grown as described by Galland et al. (1990) in a controlled culture room at 22~ under continuous illumination from fluorescent lamps (3 W - m-Z). A current of air was filtered through several stages of sterile cotton or glass-wool filters, and bubbled through the cultures. The growth period lasted some 8 d until the cultures had reached a density of approx. 106 cells-ml -a.
Isolation procedure. The flagella were isolated according to the method of Gualtieri et al. (1986) with modifications by Galland et al. (1990). In a first low-speed centrifugation (1000 - 9 for 10 rain) at room temperature, the cells were pelleted and then resuspended at a titer of about 107 cells per ml. (Higher accelerations or chilling cause premature deflagellation.) All subsequent steps were carried out at 4 ~ C or on ice. Calcium chloride was added to a final concentration of 100 mM to detach the flagella which were then separated by a second lowspeed centrifugation (1000 9g for 10 min at 4 ~ C) to sediment the cells, and finally pelleted at 22 000 9g for 20 min. The flagella were resuspended in 2 ml binding-assay buffer (10 mM Tris-HC1, 5 mM MgSO4, pH 7.5), blended in a glass homogenizer and frozen at -80~ for later tests. Freezing did not affect the binding capacity. Cells were homogenized by grinding them to powder under liquid N 2 in a mortar. In one experiment (see Fig. 4), the cell homogenate was prepared by treating the material, frozen in liquid N2, for 6 - 30 s in a "MikroDismembrator II" (from Braun, Melsungen, FRG).
A. Nebenfiihr et al. : Riboflavin-bindingto Euglena flagella At all stages of the isolation, cells and-or flagella were counted under a light microscope (Type III RS; Zeiss, Oberkochen, FRG). The final yield was low, usually about 5% of the flagella present in the original culture.
Binding assay. The binding test - modified according to Hertel et al. (1980) - was routinely performed in 1.5-ml micro centrifuge tubes (Eppendorf, Hamburg, FRG) under normal laboratory light. Test buffer (10 mM Tris-HC1, 5 mM MgSO4, pH 7.5, with 0.1 or 0.2% octylglucoside), [i4C]RF (often 5. 10 -8 M) and varying amounts of unlabelled RF were added as specified. The detergent was added to facilitate ligand access. If the binding properties of reduced RF were to be investigated, 5 mM Na-dithionite was added quickly from 125 mM dithionite dissolved in H20 immediately before use. The assay was started with the addition of 100 lal of the Euglena material. The final volume was 0.5 ml per test. After an incubation period of 10 rain on ice, the total volume was diluted in 5 ml of 10 mM Tris-HC1 (pH 7.5) and immediately poured over glass-fiber filters (Whatman GF/B; Bender und Hobein, Freiburg, FRG) into a vacuum flask; the filters had been preincubated in 0.3% polyethyleneimine (PEI) for 1 h (filter test modified after Thein and Michalke 1988). To avoid unspecific attachment of radioactivity to the filters they were rinsed with another 5 ml of 10 mM Tris-HC1 (pH 7.5). The PEI-coated filters, where the protein-RF-complex is retained, were clear of liquid about 10 s after dilution, and were then transferred to scintillation fluid (Ultima Gold from Beckmann Co, Mfinchen, FRG) and counted in a Beckmann Liquid Scintillation Counter. 'Saturable' or 'specific' [14C]RF binding is defined as the difference in filter-bound radioactivity (in cpm) between a sample containing only radioactive RF and a parallel sample to which saturating amounts of unlabelled RF had been added. Binding is often expressed as % binding=saturably bound cpm- 100/total cpm in assay. This value per mg protein added to the assay, can be used to compare different preparations. Routinely, assays were run in duplicate; the mean is presented. Table 1 illustrates the raw data and the unspecific binding: e.g. the duplicate samples for the left column were 85 and 75 cpm, or 13 and 35 cpm, respectively, and for the right column 212 and 214 cpm, or 14 and 13 cpm, respectively. This gives an impression of the accuracy of the binding tests. Most other data are presented as "specific (= saturable) binding".
Polyacrylamide 9el electrophores&, Western blots and prote& determination. Samples from Euglena cells and flagella were separated on a 10% acrylamide sodium dodecyl sulfate gel according to Laemmli (1970). One half of the gel was then transferred to a semi-dry blotting apparatus (Biometra, G6ttingen, FRG); the proteins were blotted electrophoretically to a nitrocellulose membrane (BA 83, 0.2 ~tm; Schleicher und Schiill, Dassel, FRG). Positive bands were detected using mouse monoclonal anti-a-tubulinantibodies (1/2000 in test) and enzyme-conjugated anti-mouse-IgG (1/3000), and the blot was stained with 5-bromo-4-chloro-3-indolyl phosphate, ptoluidine salt/nitro blue tetrazolium (BCJP/NBT). Western blotting and staining according to Harlow and Lane (1988). For protein determination the Coomassie method (Spector 1978) was used.
Results
Riboflavin binding in flagellar preparations. The first a i m was to detect a n d characterize s a t u r a b l e b i n d i n g o f [14C]RF in flagellar m a t e r i a l isolated a c c o r d i n g to G u a l tieri et al. (1986). This p r o c e d u r e e m p l o y s a c a l c i u m shock that causes the flagella to beat vigorously, t h e r e b y everting the a n t e r i o r i n v a g i n a t i o n in which the base o f the flagellum is attached. This ejection shifts the p o i n t o f
A. Nebenfiihr et al. : Riboflavin-binding to Euglena flagella highest stress towards the flagellar base, just above the cell membrane. As a consequence, flagella break off below the PEB which remains attached to its flagellum. Thus, isolation of flagella by differential centrifugation will co-purify PFBs and separate them from the cell bodies. During the procedure an enrichment of flagellar structures and of tubulin (see below, Fig. 4) was consistently observed, The flagellar structures counted under the light microscope were of very variable length. Either they broke off at different points outside the invagination, or they were broken during homogenization. Therefore, one has to assume that not all o f the flagellar pieces counted probably less than 50% - carried a P F B . . ~ The results in Table 1 show that both oxidized and reduced R F can bind to the flagella isolated as described above. In the flagellar preparations, p4C]RF binding was proportional to protein concentration, at least between 0 and 60 gg (data not shown). Dithionite indeed acts as a reducing agent. The products o f its oxidation, e.g. bisulfite, do not interfere with the test, since R F remains in the reduced state which is not attacked by bisulfite (Miiller and Massey 1969). After 40 min incubation of the dithionite test mixture, reoxidation had occurred as seen by the reappearance o f oxidized (yellow) RF. The R F binding, both in the oxidized and the reduced state, was reversible since radioactivity could be displaced f r o m the sites after the binding incubation, by adding a surplus of unlabelled R F to the dilution medium and leaving the diluted samples for 10 min (data not shown). Saturation curves with increasing R F concentrations are presented in Fig. la. F r o m these data and f r o m the corresponding Scatchard plots (Fig. lb), affinities and number o f sites can be estimated. In the absence of dithionite, the n u m b e r of "oxidized" binding sites (Fig. 1b, open symbols, intersection of the regression line with the abscissa: 3.2 pmol per assay, corresponding to 0.05 nmol 9 m g - 1 protein), and their affinity towards the ligand (Fig. l b, negative reciprocal of the slope, a K D --- 0.07 laM) were calculated. In another similar experiment these values were 0.8 pmol and 0.09 gM, respectively. In the presence of dithionite, the n u m b e r o f binding sites (Fig. lb, closed symbols) was determined as 7 pmol
67 a 120
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Reduced
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214 cpm 14 cpm 200 cpm 11 274
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O.02J Table 1. p4C]Riboflavin binding to a flagellar preparation from Euglena in the presence (reduced) and in the absence (oxidized) of dithionite. 40 gg protein or approx. 4 9107 flagella were used in each sample. The radioactive ligand had a concentration of 4- 10 -s M [14C]RF (i.e. 1861 cpm); 3 9 10-5 M unlabelled RF was added to part of the samples as competitor. Each value is based on duplicate determinations
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Fig. 1. a Saturation curves at increasing RF concentrations under oxidizing conditions (open symbols), using 60 gg of protein (approx. 6 - 107 flagella) in each test sample, and under reducing conditions (5 mM dithionite added; closed symbols), using 6.9 gg protein (4.2 - 106 flagella) b Scatchard plots calculated from data in a under oxidizing conditions (open symbols), and under reducing conditions
(closed symbols)
A. Nebenfiihr et al. : Riboflavin-bindingto Euglena flagella
68
E
underestimated because the flavins present in vivo might still be occupying some of the sites.
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Fig. 2. Saturation curves with increasing concentrations of R F (squares), F M N (triangles), and F A D (diamonds). Each sample contained 40 lag protein, approx. 4 - 106 flagella. To this was added 4.1 9 10 -8 M (i.e. 2000 cpm) [14C]RF and varying concentrations of unlabelled flavins, but no dithionite. The values at 10-7 M (filled symbols) represent the means of four replicates each; here, the standard error were about the size of the symbols (___2 cpm)
per assay (corresponding to 1.0 nmol. mg -1 protein), and their R F affinity was characterized by a dissociation constant K[, of 0.7 rtM. Centrifuge binding assays (according to Hertel et al. 1980) produced essentially the same results as did the filter tests (data not shown). The binding tests were usually performed in laboratory daylight. Control experiments under red safelight did not reveal any difference from those under the standard conditions (data not shown), i.e. there was no effect of light on RF binding. To determine whether RF was a specific ligand for these sites, increasing concentrations of some analogs were tested for their capacity to compete with [x4C]RF (Fig. 2). When tested in the absence of dithionite, FAD showed a much lower affinity for the binding sites, whereas F M N was more effective in replacing p4C]RF. However, it still had significantly less affinity than RF itself, by a factor of approx. 2. The same pattern was obtained in the reduced state; substances like xanthopterin and indoleacetic acid did not compete at all (data not shown).
Estimate of number of binding sites per flagellum. The molarity of RF-binding sites in the assays can be converted to binding sites per protein or - more relevantly - the number of binding sites per flagellar piece counted. In experiments without dithionite approx. 3" 104 sites per flagellum are estimated. In the case of the reduced ligands (see Fig. lb, closed symbols), however, the number of binding sites seems to be much higher, approx. 5 9 105 per flagellum. (It should be noted that in six separate and independent experiments, the estimated number of binding sites per flagellum varied by a factor of 2.8.) The estimates of binding sites represent minimal values only because the number of "entire" flagella may be overestimated. On the other hand, the number of sites could be
Localization of binding sites. The main experimental aim was to compare flagella and cell bodies of Euglena with respect to RF binding. The isolation procedure according to Gualtieri et al. (1986), separates flagella with intact PFBs from the cells; thus, the presumed photoreceptor and - according to our hypothesis - the RF-binding sites should be highly enriched in flagella compared to whole cells. In numerous experiments, using independent preparations, and under various conditions, the RF-binding capacity of isolated flagella as well as that of the cells, was assayed. The most striking and important qualitative feature in all independent tests was the high enrichment of in vitro RF binding per unit of protein in the flagella when compared to cells. Saturation curves of RF binding to flagella and to deflagellated cells were compared under oxidizing conditions (Fig. 3). The absence of binding capacity in the cell homogenate might be attributed to the action of substances present in this crude preparation and inhibiting RF binding. However, the possibility of such an inhibitor was ruled out since a mixture of the isolated flagella and the cell homogenate showed exactly the same binding capacities as did the flagella alone (Fig. 3). For reducing conditions, the same difference between flagella and cells was found (Table 2: one experiment in detail; Table 3: summary of three tests). In these experiments (Tables 2, 3), isolated flagella were compared to a homogenate of still-flagellated cells. These cells did show RF-binding capacity which was small on a protein basis, but the numbers per cell are similar to those per flagellum (Table 2, last line). Again, the strong enrichment of binding per protein in flagella cannot be attributed to inhibitor(s) in the cell 90 80~-~ 70(D
~
6o
~ 5o o
~ 40 5
30
o 20 o
"~ 10 ~
0
'
0-8
'
'
' ' ' " 1
10 -7
Riboflavin
'
'
'
' ' ' " 1
'
'
. . . . .
10 -6 concentration
'l
'
10 -'
'
'
' ' ' "
10-*
[tool-l-']
Fig. 3. Riboflavin binding to flagella and cell homogenates of Euglena, and mixtures thereof. The flagella and cell samples contained 30 gg and 40 lag protein, respectively. The mixtures were simple additions of 30 lag flagellar and of 40 gg cell protein. Other conditions were as in Fig. 2. (D-[] flagella, V-V cell homogenate, /x-/x flagella + cell homogenate)
A. Nebenfiihr et al. : Riboflavin-binding to Euglena flagella
69
Table 2. Binding of R F to isolated flagella and cell homogenates of Euglena and to a mixture thereof. 6 . 5 - 1 0 -8 M (i.e. 3048cpm) [14C]RF 4-3 - 10 -5 M unlabelled R F was added. All samples contained 5 m M Na-dithionite to achieve reducing conditions. 'Pieces' are either ceils, or flagella, or fragments of flagella counted in the microscope. Numbers in parentheses are extrapolated for the mixture Homogenate Flagella from cells + homogenate from cells
Flagella
Protein (pg) N u m b e r of pieces
6.0 4.9 9 106
cpm spec. bound % bound/free R F % b o u n d / f r e e - m g -x protein Binding sites/piece
67.4 2.6 9 106
(73.40) (7.5 9 106)
29.0
26.9
51.2
0.95 158.6
0.88 13.1
1.68 22.9
4.3 - 105
7.4" l0 s
(4.9" 105)
Table 3. Binding of R F to flagella and cell homogenates of Euglena and to mixtures thereof: three independent preparations. The cells used still carried their flagella. The protein concentration of the cell homogenate was usually about ten times that of the flagellar sample. All samples contained 5 m M Na-dithionite to obtain reducing conditions. The values are given as '% b o u n d / f r e e ' m g -1 protein'. Other conditions as in Table 2 Expt. No.
R F binding in flagella
1 2 3
159 171 154
homogenates from cells
fagella + homogenates from cells
13 8 11
23 18 13
where flagella had been shaken off (Table 4). As another control, a "flagellar" preparation was included which was derived from cells that were previously deprived of their flagella; its binding capacity was very low (Table 4, third column). In some experiments the flagellar content of the preparations was checked by immunodetection of tubulin in the different samples. For this we employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots with subsequent incubation with anti-tubulin antibodies. Euglena tubulin antigen appeared at the expected molecular weight of 55 kDa, and co-migrated with known bovine brain tubulin (data not shown). Figure 4 demonstrates the relative abundance of tubulin in the flagellar preparation compared to the cells. The RF binding (oxidized state) in these two fractions was determined to be 16.2% 9rag- 1 protein for flagella, and 2.0 % 9mg-1 protein for cells. The result confirms the microscopic estimates and again shows that RF binding is enriched in the flagella. In this experiment, the cells were homogenized with an additional vigorous treatment in a "micro-dismembrator". The resulting extract was taken up in nine volumes of test buffer, and large pieces and cells were removed by a centrifugation at 1300"9 for 10 min. This preparation showed the same binding properties as did the standard cell homogenate used in the other experiments.
Riboflavin binding to flagellar preparations and their supernatant. During this study, it was found that the
homogenate because the absolute amount of saturably bound radioactivity in the mixture of flagella and cells is the sum of the values for flagella, and for cells alone (Table 2). It should be noted that the binding values per unit of protein in Tables 2 and 3 are low for the mixtures because binding sites are "diluted" by the large amount of cell protein added. It was also found that homogenates from intact Eug~ lena cells display better RF binding than those from cells
supernatant obtained from a flagellar preparation contained an appreciable number of RF-binding sites. Flagella were sedimented at the intermediate centrifugal force of 22 000"g (characterization and R F binding shown in Table 5, left column); the resulting supernatant was subjected to a further, high-speed centrifugation at 200 000"g for 40 min. This final pellet showed RF binding (Table 5, right column), and it contained flagella, but in numbers too small to account for the large amount of binding observed. If, during preparation of flagella, some PFBs - much smaller than flagella - were broken off they might be left in the supernatant of the centrifugate at 22 000 9g, sedimenting, however, at the higher force.
Table 4. Binding of R F in various flagellar and cellular preparations. Deflagellated Euglena cells were obtained as described in Materials and methods. The 'flagellar' preparation in the third colu m n was derived from previously deflagellated cells by a second Ca 2+ shock, corresponding to fraction III in Galland
et al. (1990), (Fig. 1 .). All samples contained 40 lag protein, except for that in the third column with only 18 lag, and a very low number of flagella. No dithionite was added. Each sample contained 3 9 10 -8 M [14C]RF; 3 - 10 -5 M unlabelled R F was added to half of the samples
Starting from whole cells :
deflagellated cells:
flagella
homogenate
'flagella'
homogenate
+ Unlabelled R F Saturable binding
94 cpm 22 cpm 72 cpm
58 cpm 26 cpm 32 cpm
34 cpm 23 cpm 11 cpm
32 cpm 20 cpm 12 cpm
% bound/free R F % bound/free 9 mg -1 protein
3.9 96.3
1.7 42.8
0.6 32.7
0.6 16.0
A. Nebenfiihr et al. : Riboflavin-binding to Euglenaflagella
70 M
F5
FIO
C5
C10
M
ii !! ii i~i
55 k D a >
Table 5. Binding of RF to isolated flagella of Euglena and to the high-speed pellet from a 'flagellar' supernatant (centrifugation at 200000 9g for 40 min). The [14C]RF concentration was 7 9 10 -8 M. All samples contained 5 m M Na-dithionite. The number of binding sites was calculated assuming a KD of 0.7 taM
Protein (gg) Number of flagella Saturable binding (cpm) Bound/free 9mg -l protein (%) Binding sites in assay Binding sites/flagellum
Flagella
Pelleted supernatant
6.9 4.2 l06 39.2 170.8 2.6.1012 6.2.105
6.9 1.6 " 106 39.3 171.0 2.6.1011 1.6- 106
'
Furthermore, preliminary tests indicate that flagella without a PFB bind much less RF. The isolated flagella had been subjected to an additional Ca 2 + shock. After a subsequent centrifugation, according to Brodhun and Hfider (1990), the pelleted flagella should contain no PFBs. In parallel assays, both regularly prepared flagella and flagella with their PFBs removed were exposed to [14C]RF: in the oxidized state, the latter showed a saturable R F binding (as bound/free 9 m g - 1 protein) of only 0.21 as compared to 0.96 for control flagella. M
F5
FIO
C5
C10
M
Discussion
Fig. 4. a Analysis of flagella and cell preparations by SDS PAGE,
and b subsequent Western blot with anti-a-tubulin antibodies. The proteins in a and b were separated on one common 10% gel which subsequently was cut into two halves for protein staining and blotting, respectively. The lanes were loaded with 5 tag and 10 tag protein, respectively, as indicated above each lane. F, flagellar preparation; C, cell extract; M, molecular-weight markers (prestained markers from Sigma, FRG)
Proteins, in the plasma m e m b r a n e or in another "fixed" position, that bind R F have been proposed as blue-light photoreceptors in fungi and plants (Hertel 1980). The results here document the occurrence of a strong and saturable binding of R F to flagellar preparations from Euglena. Both oxidized and reduced R F can bind specifically, i.e. better than F M N or F A D . Affinity for reduced as well as for oxidized R F was reflected by Ko values in the range of 0.1 - 1 gM. The number of RF-binding sites seems to be higher in the presence of dithionite. The difference between the number o f sites in the reduced and the oxidized state suggests that one is dealing with two different types of binding proteins. The decision as to whether there are two or more different binding sites involved, or just different affinity patterns at one site only, must await isolation and characterization of the respective proteins. Such a study is presently under way. F r o m the a m o u n t of R F binding and from the number of flagella (or flagellar pieces) in the assay, approx. 5" 105 RF-binding sites per flagellum were estimated under reducing conditions. The amount per PFB might actually be higher by a factor of two, or more, since probably only half of the "flagella" counted carried their PFB. The number of 106 binding sites per PFB should be compared to: (i) the a m o u n t of binding sites from one intact cell (Table 2); (ii) the number of photoreceptor molecules in the PFB that can be expected from microspectrophotometric observations (Colombetti and Lenci 1980); (iii) the number of flavin molecules in/at the PFB determined by Colombetti and Lenci (1980) by microspectrofluorimetry; and (iv) the number of unit cells o f the PFB's protein crystal (Piccini and M a m m i 1978).
A. Nebenfiihr et al. : Riboflavin-binding to Euglena flagella Since all these n u m b e r s are in the range o f approx. 106, it seems reasonable to p r o p o s e that the R F - b i n d i n g sites are localized in the P F B or at the s u r r o u n d i n g m e m b r a n e , and that these proteins are involved in p h o t o r e c e p t i o n for euglenoid phototaxis. It has been observed that the flavin-type fluorescence in the P F B region o f Euglena described by Benedetti and Checcucci (1975) a n d b y Schmidt et al. (1990) is lost when preparing and washing flagella (Galland et al. 1990, fraction II in their Fig. 1). These findings agree very well with a "loose" flavin binding to the flagellar structures. The finding by B r o d h u n and H/ider (1990) o f some covalently (or tightly) b o u n d flavin in flagellar preparations is n o t incompatible with the large a m o u n t o f reversible R F binding s h o w n here. Gualtieri et al. (1989) m e a s u r e d a b s o r p t i o n spectra f r o m a single "dried" P F B in situ. The a m o u n t o f pigment m u s t be very high, in the range o f 107 molecules per PFB. The interpretation o f the spectrum as being that o f a r h o d o p s i n is n o t in line with the reported action spectra (see Introduction). We rather suggest that P F B s contain a protein-stabilized " r e d " flavin semiquinone radical (see Massey a n d Palmer 1966, where e.g. D-amino acid oxidase shows a spectrum like the one m e a s u r e d by G u a l tieri et al. 1989). The pterins - p r o b a b l y present at the P F B - m a y act as a second p h o t o r e c e p t o r , or as a c o f a c t o r for the p h o t o reactions ( B r o d h u n a n d H~ider 1990; G a l l a n d et al. 1990; Schmidt et al. 1990). Interference o f pterins with R F binding, however, was n o t seen when x a n t h o p t e r i n was added. The determination o f the m e c h a n i s m o f p h o t o p e r c e p tion in Euglena is o f general interest because this p r o t o zoon/phytoflagellate m a y be at the base o f eukaryotic evolution. Euglena's - a n d m o s t protists - precise phylogenetic relationships are far f r o m clear, but it m i g h t actually represent a p r o t o z o o n that carries a e u k a r y o t i c alga as an e n d o s y m b i o n t . F o r example, its pellicle lacking cellulose is atypical o f cell walls o f green algae, and the "chloroplasts" have one additional m e m b r a n e which has been interpreted as the r e m n a n t o f an e n d o c y t o t i c event in which a e u k a r y o t i c green alga was i n c o r p o r a t e d into the pre-Euglena cell (Gibbs 1978). In view o f these relationships it is m o s t interesting that other non-green algae also s h o w a strong flavin-like fluorescence in one o f their flagella (Kawai 1988; Colem a n 1988). Riboflavin-binding sites in these flagella w o u l d be a n o t h e r strong a r g u m e n t for a p h o t o p h y s i o l o g ical role o f such flavoproteins. It should be noted, h o w ever, that in Euglena, flavin fluorescence o f the flagellum - in c o n t r a s t to the P F B - is very weak (Kawai 1988). This work was supported by the Deutsche Forschungsgemeinschaft
References Beale, S.I., Foley, T., Dzelzkalns, V. (1981) 8-Aminolevulinic acid synthase from Euglena graeilis. Proc. Natl. Acad. Sci. USA 78, 1666-1669 Benedetti, P.A., Checcucci, A. (1975) Paraflagellar body (PFB) pigments studied by fluorescence microscopy in Euglena graeilis. Plant Sci. Letts. 4, 47-51 Brodhun, B., H~ider, D.P. (1990) Photoreceptor proteins and pig-
71 ments in the paraflagellar body of the flagellate Euglena 9racilis. Photochem. Photobiol. 52, 865-872 Checcucci, A., Colombetti, G., Ferrara, R., Lenci, F. (1976) Action spectra for photoaccumulation of green and colorless Euglena: evidence for identification of receptor pigments. Photochem. Photobiol. 23, 51-54 Coleman, A.W. (1988) The autofluorescent flagellum: a new phylogenetic enigma. J. Phycol. 24, 118-120 Colombetti, G., Lenci, F. (1980) Identification and spectroscopic characterization of photoreceptor pigments. In: Photoreception and sensory transduction in aneural organisms (NATO ASISeries A 33), pp. 173-188, Lenci, F., Colombetti, G., eds. Plenum Press, New York Colombetti, G., Lenci, F., Diehn, B. (1982) Responses to photic, chemical, and mechanical stimuli. In: The biology of Euglena III. pp. 169-196, Buetow, D.E., ed. Academic Press, New York Dohrmann, U. (1983) In vitro riboflavin binding and endogenous flavins in Phycomyces blakesleeanus. Planta 159, 357-365 Fritz, B.J., Kasai, S., Matsui, K. (1989) Free cellular riboflavin is involved in phase shifting by light of the circadian clock in Neurospora crassa. Plant Cell Physiol. 30, 557-564 Galland, P., Senger, H. (1988) New trends in photobiology: The role of flavins as photoreceptors. J. Photochem. Photobiol. B 1, 277-294 Galland, P., Keiner, P., D6rnemann, D., Senger, H., Brodhun, B., H~ider, D.P. (1990) Pterin- and flavin-like fluorescence associated with isolated flagella of Euglena 9racilis. Photochem. Photobiol. 51, 675-680 Gibbs, S.P. (1978) The chloroplasts of Euglena may have evolved from symbiotic green algae. Can. J. Bot. 56, 2883-2889 Gualtieri, P., Barsanti, L., Rosati, G. (1986) Isolation of the photoreceptor (paraflagellar body) of the phototactic flagellate Euglena 9racilis. Arch. Microbiol. 145, 303-305 Gualtieri, P., Barsanti, L., Passarelli, V. (1989) Absorption spectrum of a single paraflagellar swelling of Euglena 9racilis. Biochim. Biophys. Acta 993, 293-296 Harlow, E., Lane, D. (1988) Antibodies: a laboratory manual. Cold Spring Harbor Lab., New York Hertel, R. (1980) Phototropism in lower plants. In : Photoreception and sensory transduction in aneural organisms (NATO ASISeries A 33), pp, 89-105, Lenci, F., Colombetti, G., eds. Plenum Press, New York Hertel, R., Jesaitis, A.J., Dohrmann, U., Briggs, W.R. (1980) In vitro binding of riboflavin to subcellular particles from maize coleoptiles and Cucurbita hypocotyls. Planta 147, 312-319 Kawai, H (1988) A flavin-like autofluorescent substance in the posterior flagellum of golden and brown algae. J. Phycol. 24, 114-117 Kuznicki, L., Mikolajzcyk, E., Walne, P.L. (1990) Photobehavior of euglenoid flagellates: theoretical and evolutionary perspectives. Crit. Rev. Plant Sci. 9, 343-369 Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680-685 Massey, V., Palmer, G. (1966) On the existence of spectrally distinct classes of flavoprotein semiquinones. Biochemistry 5, 3181-3189 Miiller, F., Massey, V. (1969) Flavin-sulfite complexes and their structures. J. Biol. Chem. 224, 4007-4016 Piccini, E., Mammi, M. (1978) Motor apparatus ofEuglena 9racilis: ultrastructure of the basal portion of the flagellum and the paraflagellar body. Boll. Zool. 45, 405-414 Presti, D.E., Hsu, W.J., Delbriick, M. (1977) Phototropism in Phyeomyees mutants lacking [3-carotene. Photochem. Photobiol. 26, 403-405 Schl6sser, U.G. (1982) Sammlung von Algenkulturen. Ber. Dtsch. Bot. Ges. 95, 181-276 Schmidt, W., Galland, P., Senger, H., Furuya, M. (1990) Microspectrophotometry of Euglena gracilis. Pterin- and flavin-like fluorescence in the paraflagellar body. Planta 182, 374-381 Spector, T. (1978) Refinement of the Coomassie-Blue-method of protein quantitation. Annal. Biochem. 86, 142-146 Thein, M., Michalke, W. (1988) Bisulfite interacts specifically with binding sites of the auxin transport inhibitor N-1-Naphthylphthalamic acid. Planta 76, 343 350
